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The story begins September 1941 just before the attack on Pearl Harbor. Shy bookkeeper Henry Limpet loves fish with a passion. When his friend George Stickle enlists in the United States Navy, Limpet attempts to enlist as well, but is rejected. Feeling downcast, he wanders down to a pier near Coney Island and accidentally falls into the water. Inexplicably, he finds he has turned into a fish. Since he never resurfaces, his wife, Bessie, and George assume he has drowned.

The fish Limpet, complete with his signature pince-nez spectacles, discovers a new-found ability during some of his initial misadventures, a powerful underwater roar, his "thrum". He falls in love with a female fish he names Ladyfish, the concept of names being unknown to her. He later makes friends with a misanthropic hermit crab named Crusty.

Still determined to help the Navy, Limpet finds a convoy and requests to see George. With George's help, Limpet gets himself commissioned by the Navy, complete with advancing rank and a salary, which he sends to Bessie. He helps the Navy locate Nazi U-boats by signaling with his "thrum", and plays a large part in the Allied victory in the Battle of the Atlantic. In his final mission, he is nearly killed when the Nazis develop a "thrum" seeking torpedo, and is further handicapped by the loss of his spectacles. He manages to survive using Crusty as his "navigator", and sinks a number of U-boats by redirecting the torpedoes. After the battle, he swims to Coney Island to say goodbye to Bessie (who has now fallen in love with George) and gets a replacement set of glasses. He then swims off with Ladyfish.

In the film's coda, set in the modern times of 1964, George (now a high ranking naval officer) and the Admiral are presented with a report that Mr. Limpet is still alive and working with porpoises. The two men travel out to sea to contact Mr. Limpet and offer him a commission in the United States Navy. It is unknown what became of the conversation, for the movie ends with a question mark.

The film had its premiere on January 20, 1964 at the Weeki Watchee Underwater Theater in Spring Hill, Florida. It was the world's first underwater movie premiere. The film went into general release on March 28, 1964.

The film had its television premiere on CBS on December 29, 1968, as part of The CBS Friday Night Movies.

During World War I and World War II, there was a mine known as a limpet, a type of naval mine attached to a target by magnets named because of their superficial similarity to the limpet, a type of mollusk. "Das Limpet" was the German Navy's identification of Don Knott's character.

The USS Alfred A. Cunningham was the naval ship featured in this film.
Another ship used in filming was the USS Galveston, which was referred to as the USS Los Angeles in the film. The Los Angeles was offered for use at the time of pre-production planning, but was decommissioned in the fall of 1963, before principal filming began. Here lies a double anachronism, in that the Los Angeles was not commissioned until the fall of 1945, and the Galveston had been converted to a guided missile cruiser, and clearly shows her 1960's configuration with large radars and missile launchers in place of her removed gun turrets.